Method of preparing hepatitis B core antigen

ABSTRACT

HB c  Ag is prepared by isolating Dane particles by isopycnic banding of fluid from HB s  Ag positive donors, optionally but preferably pelleting the Dane particles, and then removing the surface antigen by contacting the Dane particles with a nonionic surfactant having from about 15 to about 35 oxyethylene units in the presence of a reducing agent such as mercaptoethanol. The isolated core antigen is used as a diagnostic and immunologic agent.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to hepatitis B core antigen (HB_(c) Ag) and, moreparticularly, to a process for preparing hepatitis B core antigen inhigh yield and purity.

Hepatitis B is one of the types of viral hepatitis which results in asystemic infection with the principal pathologic changes occurring inthe liver. This disease affects mainly adults and is maintained chieflyby transfer of infection from long term carriers of the virus. Usualmethods of spread are by blood transfusion, contaminated needles andsyringes, through skin breached by cuts or scratches, by unsterilizeddental instruments as well as by saliva, veneral contact or exposure toaerosolized infected blood.

The incubation period of type B hepatitis is relatively long: from 6weeks to 6 months may elapse between infection and the onset of clinicalsymptoms. The illness usually begins with fatigue and anorexia,sometimes accompanied by myalgia and abdominal discomfort. Laterjaundice, dark urine, light stools and tender hepatomegaly may appear.In some cases, the onset may be rapid, with appearance of jaundice earlyin association with fever, chills, and leukocytosis. In other casesjaundice may never by recognized and the patient may be aware of a"flu-like" illness. It is estimated that the majority of hepatitisinfections result in a mild, anicteric illness.

Serum obtained from patients with hepatitis B infections usuallycontains three distinct morphologic forms. The largest of thesemorphologic forms, a 42-nm to 45-nm double shelled spherical particle,often referred to as the Dane particle (HBV), is believed to be thevirus of hepatitis B. The outer surface or envelope of the Dane particle(HB_(s) Ag) surrounds a 27-nm inner core which does not react withantibody against HB_(s) Ag and which contains a distinct antigen, thecore antigen (HB_(c) Ag). Antibody to HB_(c) Ag appears after acutehepatitis B infection, and also can be demonstrated consistently inchronic carriers of HB_(s) Ag. Highly sensitive techniques are nowavailable for detection of the HB_(c) Ag system. A deterrent to the morewidespread use of such techniques, however, is the absence of a simpleyet practical and effective method for obtaining HB_(c) Ag. The methodsproposed heretofore generally involve the use of selected plasma whichcontains exceptionally high amounts of Dane particles.

2. Objects of the Invention

It is, accordingly, an object of the present invention to provide apractical and effective method for obtaining HB_(c) Ag. Another objectis to provide an improved method for concentrating and purifying HB_(c)Ag. Still another object is to provide a method for obtaining HB_(c) Agfrom biological fluid found positive for HB_(s) Ag rather than fromselected high titer HB_(s) Ag plasma. A further object is to provideimmunogenic and therapeutic compositions containing HB_(c) Ag. These andother objects of the present invention will be apparent from thefollowing description.

SUMMARY OF THE INVENTION

HB_(c) Ag is prepared by isolating Dane particles by isopycnic bandingof biological fluid from human HB_(s) Ag positive donors, optionally butpreferably pelleting the Dane particles, and then removing the surfaceantigen by contacting the Dane particles with a nonionic surfactanthaving from about 15 to about 35 oxyethylene units in the presence of areducing agent such as mercaptoethanol.

DETAILED DESCRIPTION

The starting material for the purified hepatitis B core antigen (HB_(c)Ag) of the present invention is plasma obtained from donors positive toHB_(s) Ag. The plasma is obtained in conventional manner, e.g., byplasmaphoresis. The level of HB_(s) Ag may be measured in known mannerby any suitable means, e.g., reversed passive hemagglutination orcomplement fixation. Optionally, the plasma may be cooled and thecryoprecipitate which forms is removed by light centrifugation. The Daneparticles in the resulting plasma are isolated by isopycnic banding. TheDane particle-rich fraction is treated to remove Dane core antibody,preferably by pelleting, and then treated to remove the surface antigenand liberate the core antigen. Removal of the surface antigen iseffected by contacting the Dane particle with a nonionic surfactanthaving from about 15 to about 35, preferably about 18 to about 33,oxyethylene units in the molecule in the presence of a mercaptanreducing agent, for example, mercaptoethanol, dithiothreitol,dithioerythritol, and dithiooctanoic acid. Suitable nonionic surfactantsare oxyethylated alkyl phenols, polyoxyethylene sorbitan fatty acidesters, polyoxyethylene acids, polyoxyethylene alcohols, polyoxyethyleneoils and polyoxyethylene oxypropylene fatty acids. Some specificexamples are the following:

alkylphenoxypolyethoxy (30) ethanol

polyoxyethylene (20) sorbitan monolaurate

polyoxyethylene (20) sorbitan monopalmitate

polyoxyethylene (20) sorbitan monostearate

polyoxyethylene (20) sorbitan tristearate

polyoxyethylene (20) sorbitan monooleate

polyoxyethylene (20) sorbitan trioleate

polyoxyethylene (20) palmitate

polyoxyethylene (20) lauryl ether

polyoxyethylene (20) cetyl ether

polyoxyethylene (20) stearyl ether

polyoxyethylene (20) oleyl ether

polyoxyethylene (25) hydrogenated castor oil

polyoxyethylene (25) oxypropylene monostearate.

In isopycnic banding the partially purified concentrate is contactedwith a liquid medium having a density gradient therein which includesthe density of the specific antigen being isolated. The liquid medium isthen subjected to ultracentrifugation to attain an equilibriumdistribution of the serum components through the density gradientaccording to their individual densities. Successive fractions of themedium are displaced and those containing the desired antigen, i.e. thefractions having a density of from about 1.26 to about 1.30 g/cc, areseparated. The concentrations of the solutions forming the gradient areselected so as to encompass the density range of from about 1.0 to about1.41 g/cc. The liquid medium may be employed in the form of a lineargradient or a step gradient. Preferably it is employed in the form of astep gradient due to its inherently higher capacity for fractionation.

The liquid media used in the isopycnic banding step may be any densitygradient in the appropriate ranges, e.g. sucrose, potassium bromide,cesium chloride, potassium tartrate, or sodium bromide. Sodium bromideis preferred.

EXAMPLE 1 A. Preparation of Dane Particles (HBV)

The rotor of a centrifuge, Electronucleonics K is filled with 8,400 mlof phosphate buffer. After running the rotor up to 10,000 rpm to degasthe system, the following step gradient is pumped into the bottom of thestationary rotor:

1. 2,400 ml of 10% NaBr, ρ = 1.08

2. 1,000 ml of 20% NaBr, ρ = 1.17

3. 1,500 ml of 30% NaBr, ρ = 1.28

4. 3,500 ml of 40% NaBr, ρ = 1.41

Plasma containing HB_(s) Ag, 1,750 ml, is pumped into the top of thestationary rotor displacing 1,750 ml of 40% NaBr from the bottom of therotor. The rotor is accelerated to 30,000 rpm and run at this speed for4 hours. The rotor is then stopped and 1,750 ml of 40% NaBr are pumpedinto the bottom of the rotor forcing the plasma out the top. Anadditional 1,750 ml of fresh plasma containing HB_(s) Ag are pumped intothe top of the rotor displacing an equal volume of 40% NaBr out thebottom of the rotor. The rotor is then run at 30,000 rpm for 18 hours.After stopping the rotor 1,000 ml of Dane particle rich material in the1.26-1.30 density region is collected.

The Dane particles (HBV) are separated from the NaBr zonal fraction inthe following procedure. The zonal fraction (1000 ml) is diluted to 3000ml using phosphate buffered saline. This material is then placed intotwelve type 19 rotor plastic bottles (ea. 250 ml/bottle). The materialis then centrifuged using a type 19 rotor (Beckman). The rotor is spunat 17,000 rpm (45,000 × g) for 24 hours in order to pellet the Daneparticles. The rotor is then stopped and the supernate from each bottleis decanted. The pellet material from all 12 bottles is recovered in atotal volume of 5-7 ml of Tris-saline buffer and stored at -70° C. Thismaterial is the Dane particle concentrate.

B. Purification of Dane Particles

1 ml of concentrated Dane particles from part A is layered over 4 ml of20% sucrose - 1% bovine serum albumin (BSA) in Tris buffer (pH 7.6) in aSW 65 rotor with 1/2 × 2 inch cellulose nitrate tubes. The particles arecentifuged at 35,000 rpms for 4 hours. Post centrifugation, thesupernate fluid is decanted and the pellet is gently resuspended in 0.5ml of Tris buffer with 1% BSA using a cotton tipped swab (pre-moistenedwith buffer). The cotton swab is then rinsed with 0.5 ml of buffer. Thefinal volume of Dane particle material is 1 ml. The Dane particles arestored at -70° C.

C. Preparation of HB_(c) Ag (Core Antigen)

The material from part B, 1 ml, is added to 1 ml of a 1% (v/v) solutionof 2-mercaptoethanol is deionized water, and 1 ml of a 1% (v/v) solutionof polyoxyethylene (20) sorbitan monooleate in deionized water. Theresulting mixture is agitated gently and placed in a 37° C. water bath.After 1 hour the mixture is diluted with TMN-1% BSA (a solutioncontaining 0.08 M Tris, 0.008 M MgCl₂ and 0.14 M NaCl, and 1% BSA) usinga previously calculated quantity of diluent until if contains 32 IAHAunits per ml. The solution is then dispensed into plastic 2 ml screw-capserum tubes (0.5 ml/tube) and stored in a liquid nitrogen freezer.

EXAMPLE 2

To each of twelve 6 ml glass vials there are added 1 ml of purified Daneparticles prepared as described in Example 1, 1 ml of a 1% (v/v)solution of 2-mercaptoethanol in deionized water, and 1 ml of a 1% (v/v)solution in deionized water of the materials listed below. The mixturesare agitated gently and placed in a 37° water bath. After 1 hour theresulting mixtures are assayed for HB_(c) Ag by the immune adherencehemagglutination assay.

    ______________________________________                                        Nonionic Surfactant      IAHA Units                                           ______________________________________                                        polyoxyethylene (20) sorbitan monooleate                                                               1000                                                 polyoxyethylene (20) sorbitan monolaurate                                                              1000                                                 polyoxyethylene (23) lauryl ether                                                                      1000                                                 polyoxyethylene (20) cetyl ether                                                                       1000                                                 alkyl phenoxypolyethoxy (30) ethanol                                                                   1000                                                 alkyl phenoxypolyethoxy (9) ethanol                                                                    240                                                  alkyl phenoxypolyethoxy (40) ethanol                                                                   240                                                  polyoxyethylene (9) octaphenol                                                                         60                                                   sorbitan monolaurate     30                                                   sorbitan monostearate    30                                                   sodium dodecyl sulfate   30                                                   polyalkylaryl sulfonic acid                                                                            30                                                   ______________________________________                                    

The foregoing results show that nonionic surfactants containing 9 or 40oxyethylene units are significantly inferior to those containing from 20to 30 oxyethylene units while those without any oxyethylene units arealmost totally ineffective.

EXAMPLE 3

To a first 1 ml sample of purified Dane particles from the same lot usedin Example 2 there are added 1 ml of polyoxyethylene (20) sorbitanmonooleate, and 1 ml of 2-mercaptoethanol. A second 1 ml sample istreated similarly except substituting 1 ml of deionized water for the2-mercaptoethanol. Each sample is mixed, incubated for 1 hour at 37° andassayed for HB_(c) Ag by the immune adherence hemagglutination assay.The second sample without 2-mercaptoethanol is found to contain lessthan half the amount of HB_(c) Ag in the first sample.

EXAMPLE 4

HB_(c) Ag as prepared in Example 1 is adsorbed on alum as follows. Tenml of HB_(c) Ag (Type Ad) containing 16-32 IA units/ml are mixed with0.85 ml of 10% alum solution KAl (SO₄)₂.12H₂ O. While stirring 0.1 NNaOH is added slowly to adjust the pH to 6.8. Mixing is continued for 1hour at room temperature. The solution is then centrifuged at 1500 × gfor 10 minutes. The supernate is decanted and the pellet is resuspendedwith saline solution to the original volume (10 mls). The solution isthen mixed for 5-10 minutes prior to use as an antigen.

EXAMPLE 5

The procedure of Example 4 is employed except using 10 ml of HB_(c) Ag(Type Ay).

EXAMPLE 6

Alum antigens prepared in Examples 4 and 5 are used to make high titerHB_(c) Ab serums. The guinea pigs are divided into two groups which areused to produce the HB_(c) Ab antiserum. The first group is administeredintramuscular injections of 3 doses of 0.5 ml at monthly intervals ofthe product of Example 4. The second group is treated similarly with theproduct of Example 5. High titered hepatitis B antibody serums areproduced in each group.

EXAMPLE 7 Enzyme-linked immunosorbant assay (ELISA)

The HB_(c) Ag obtained in Example 1 is purified by centrifugation on a20-60% sucrose gradient at 200,000 × g for 2.5 hours. The HB_(c) Ag isthen assayed to determine protein content.

The HB_(c) Ag is diluted to 1 μg/ml with 0.1M carbonate buffer pH 9.7for use in the ELISA assay.

The solid phase used in the assay is a 96 well, Cooke microtiter,U-bottom, polystyrene plate.

The enzyme-conjugate is alkaline phosphatase conjugated goat anti-humanimmunoglobulin (Engvall et al.).¹ . Use level is determined bytitration.

The enzyme substrate is 0.01% p-nitrophenyl phosphate in 0.1M carbonatebuffer pH 9.8, containing 0.001M MgCl₂.

The assay method is described by E. Nassau et al.² This method is usedto detect HB_(c) Ab in plasma and serum samples.

EXAMPLE 8

The final product of Example 1 is treated under aseptic conditions with1:4000 formalin at 37° C. for 72 hours. Excess formalin is thenneutralized with sodium bisulfite. The core antigen is then adsorbed onalum by following the procedure of Example 4.

Individuals positive for HB_(c) Ab and having an HB_(c) Ab antibodytiter (as measured by the immune adherence hemagglutination assay, IAHA)of 32 IAHA units/ml or greater are administered 1 ml (40 μg) doses ofvaccine intramuscularly. Additional injections are given 1 month and 3months following the first injection. One week after the third injectionthe individuals are plasmapheresed and HB_(c) Ab titers are run on theindividual plasma using the immune adherence hemagglutination assay. Themajority of the individuals experience an increase in their HB_(c) Abtiter compared to their initial titer. Those plasmas having an antibodytiter of 2000 or higher are processed to yield gamma globulin havinghigh HB_(c) Ab titer.

EXAMPLE 9

The material from part B of Example 1, 1 ml, is added to 1 ml of a (v/v)solution of 2-mercaptoethanol in deionized water, and 1 ml of a 1% (v/v)solution of polyoxyethylene (20) sorbitan monooleate in deionized water.The resulting mixture is agitated gently and placed in a 37° C. waterbath. After 1 hour the mixture is diluted with SPGA using a previouslycalculated quantity of diluent until it contains 32 IAHA units per ml.The solution is then dispensed into plastic 2 ml screw-cap serum tubes(0.5 ml/tube) and stored in a liquid nitrogen freezer.

What is claimed is:
 1. A method of preparing hepatitis B core antigenwhich comprises treating Dane particles with a nonionic surfactanthaving from about 15 to about 35 oxyethylene units in the presence of amercaptan reducing agent.
 2. A method according to claim 1 wherein thereducing agent is mercaptoethanol.
 3. A method according to claim 1wherein the nonionic surfactant is polyoxyethylene (20) sorbitanmonooleate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene(23) lauryl ether, polyoxyethylene (20) cetyl ether or alkylphenoxypolyethoxy (30) ethanol.
 4. A method according to claim 1 whereinthe Dane particles are obtained by subjecting biological fluid of humanhepatitis B donors to isopycnic banding in a suitable density gradient.5. A method according to claim 4 wherein the density gradient is a stepgradient and the biological fluid is plasma.
 6. A method according toclaim 4 wherein the density gradient is sodium bromide.
 7. A methodaccording to claim 4 wherein the Dane particles are pelleted after theisopycnic banding and before being treated with the nonionic surfactant.